PROJECT SUMMARY Hepatitis C virus (HCV) infects over 70 million people worldwide and is a leading cause of chronic liver cirrhosis and liver cancer. Despite the success of direct-acting antivirals, control of the HCV epidemic remains challenging due to difficulties in early virus detection, the high cost of therapy, and possible reinfection after the successful treatment. An effective prophylactic vaccine would help to control the epidemic, but the enormous genetic diversity of the HCV hampers its development. Despite the high variability of the virus, 30% of HCV-infected individuals clear the viral infection by developing broadly neutralizing antibodies (bNAbs) that bind to conserved neutralizing epitopes of HCV glycoprotein E2. By stimulating the development of bNAbs by vaccination, the HCV epidemic might be stopped. We recently identified several E2 proteins from different HCV isolates that bind to germline precursors of human bNAbs, raising the possibility of vaccine development using these variants. In this proposal, we aim to use naturally-occurring E2 proteins to design immunogens that will elicit HCV-specific bNAbs in animal models. To accomplish this goal, E2 protein or virus-like particle-based immunogens will be tested in wild-type mice and non-human primates. Next, the structures of HCV-specific antibodies induced by immunization will be determined. For this, we will sort and sequence epitope-specific B cells from vaccinated animals and express antibodies from these sequences. Then, we will determine the structures of HCV-specific bNAbs alone or in complex with immunogens to elucidate the determinants of broad neutralization of HCV by vaccine-induced antibodies. The detailed analysis of the elicited antibody response will facilitate the optimization of immunogens by enhancing their specificity and selectivity. Together, the results of these aims will lead to the identification and characterization of novel vaccine candidates that elicit HCV-specific bNAbs, facilitating the development of an effective HCV vaccine. The mentoring phase of this proposal will take place in Dr. Pamela Bjorkman's laboratory at Caltech. Since there are several critical areas of the proposed project that require additional training, Dr. Andrew Flyak has assembled a mentorship committee comprising experts in structural biology (Dr. Pamela Bjorkman), B cell immunology (Dr. Michel Nussenzweig), and HCV biology (Dr. Justin Bailey). By using an interdisciplinary approach developed throughout this project and support from the mentoring committee, Dr. Flyak will begin the independent phase of this award, answering fundamental questions that hinder the development of effective vaccines against HCV, or other rapidly mutating viruses. !